The cyanine dye class has proved to be an extremely bright and versatile class of dyes in both photographic and biological applications. The addition of sulphonic acid and attachment of functionality for conjugation have allowed them to be fully exploited for biological research applications. The addition of sulphonic acids for additional water solubility and enhanced brightness has led to the dyes becoming overall neutral or negatively charged. As described in U.S. Pat. Nos. 5,268,486 and 5,486,616, the basic cyanine structure has a +1 overall positive charge e.g. 
In certain applications dyes having several positively charged atoms can be of benefit. This invention addresses that need.
The invention provides a cyanine dye having the structure 
where the dotted lines represent the carbon atoms necessary for a one ring or a two or three fused ring system with 5 or 6 carbon atoms in each ring and R3, R4, R5 and R6 attached to the rings,
X and Y. are independently elected from O, S and CR32, where R8 is C1-C4 alkyl,
n is 1, 2 or 3,
at least one of R1, R2, R3, R4, R5, R6 and R7 comprises a reactive or a functional group,
at least one of R1, R2, R3, R4, R5, R6 and R7 incorporates one to to five positively charged nitrogen or phosphorus or sulphur atoms,
any remaining R3, R4, R5 and R6 is independently selected from H, SOxe2x80x943, Cl, Br, OR9 and SR9, where R9 is C1-C10 alkyl or aryl or aralkyl,
any remaining R1 and R2 is independently selected from C1-C10 alkyl or aryl or aralkyl either unsubstituted or substituted by SOxe2x80x943,
any remaining R7 is selected from H and C1-C10 alkyl or aryl or aralkyl either unsubstituted or substituted by SOxe2x80x943,
provided that at least two positively charged atoms selected from nitrogen and phosphorus and sulphur are present in the groups R1, R2, R3, R4, R5, R6 and R7,
and provided that the first atom of R7 (through which it is linked to the rest of the molecule) is H or C.
Preferably the cyanine dye has the structure (2) 
Preferably the cyanine dyes have an overall positive charge of +2 to +6. The overall charge of the dye may be considered as the number of positively charged nitrogen (or phosphorus or sulphur) atoms minus the number of sulphonate (or carboxyl or phosphate) groups. Thus for example, a dye having 3 positively charged nitrogen atoms and 0 or 2 or 4 sulphonate groups would have an overall charge of +3 or +1 or xe2x88x921, respectively. The extent to which an atom or group is charged may depend on the pH of its environment.
Preferably a reactive or functional group is present as a structure -L-Q where L is a linker and Q is the reactive or a functional group. A reactive group of the dye can react with a functional group of a target molecule; or a functional group of the dye can react with a reactive group of a target molecule; whereby the target molecule becomes labelled by the dye. Preferably Q is a functional group selected from primary amine, secondary amine, hydrazine derivatives, hydroxylamine derivatives, and pyrazolone. Alternatively a functional group may be selected from sulphydryl, carboxyl, hydroxyl, thiophosphate, imidazole and carbonyl including aldehyde and ketone.
Preferably a reactive group is selected from succinimidyl ester, isothiocyanate, dichlorotriazine, isocyanate, haloacetamide, maleimide, sulphonyl halide, acid halide, alkylimido ester, arylimido ester, carbodiimide, phosphoramidite, anhydride and acyl azide.
By virtue of these functional and reactive groups, the cyanine dyes of the present invention are combined with target materials to form conjugates. Suitable target materials may include antibodies, antigens, proteins, carbohydrates, lipids, nucleotides, nucleic acids, polymer particles or glass beads. Thus for example, cyanine dyes having the preferred functional groups mentioned above are suitable for reacting with carbohydrates to form conjugates therewith.
L is a linker, which may contain 1-60 chain atoms selected from C, N, O, S and P, e.g. a straight chain of 1-30 carbon atoms in which are incorporated one or more N, O, S or P atoms. For example the linker may be
"Parenopenst"CH2"Parenclosest"x 
"Parenopenst"CH2)Pxe2x80x94Oxe2x80x94(CH2)q"Parenclosest"y 
"Parenopenst"CH2)Pxe2x80x94CONHxe2x80x94(CH2)q"Parenclosest"y 
"Parenopenst"CH2)Pxe2x80x94Arxe2x80x94(CH2)q"Parenclosest"y 
where x is 1-30, preferably 1-10,
p is 1-5,
q is 0-5 and
y is 1-5.
Present in the cyanine dye of the invention is a branched or straight chain incorporating 1-5 positively charged nitrogen or phosphorus or sulphur atoms. (Some or all of these positively charged N or P or S atoms may be present in the linker group L.). Preferably each positively charged atom is a nitrogen atom provided by a quatemary ammonium group, or alternatively by a protonated tertiary amino group, a guanidinium group, an imidazole group or a pyridinium group. Positively charged P and S atoms may be provided by phosphonium ions and sulphonium ions respectively. Preferably a branched or straight chain incorporating one to five positively charged nitrogen atoms is up to 60 chain carbon atoms and has the structure
xe2x80x94(CH2)mN+R10R10R11 or
xe2x80x94CH2xe2x80x94Phxe2x80x94N+R10R10R11 
where m is 1 to 4,
R10 is C1-C10 alkyl,
and R11 is C1-C10 alkyl or xe2x80x94(CH2)mN+R10R10R11.
Or the linker group L and/or the chain incorporating positively charged nitrogen atoms may comprise one or more natural or artificial amino acid residues. It is a simple matter to introduce any number e.g. 1-20 of lysine residues, and if desired to quaternise the amino groups. Such linkers may contain the grouping xe2x80x94(CO.NHW)1xe2x80x94 where r is preferably 1 to 6 and W is aminoalkyl or quaternised aminoalkyl such as xe2x80x94(CH2)4NH2 or xe2x80x94(CH2)4N+R310 where R10 is C1-C10 alkyl.
At least two positively charged nitrogen or phosphorus or sulphur atoms and preferably at least one reactive or functional group are present in pendant groups attached to the core structure of the dye. They may be positioned on the same group or different groups R1, R2, R3, R4, R5, R6 and R7.
Preferably the cyanine dye has the structure (2)
wherein X and Y are C(CH3)2,
n is 1 or 2,
R1 is xe2x80x94(CH2)5xe2x80x94COOH,
R2 is xe2x80x94(CH2)3xe2x80x94N+(CH3)2xe2x80x94(CH2)3xe2x80x94N+(CH3)2(C2H5),
and R3 and R4 are H.
The dyes described in the experimental section below have quaternary ammonium ions attached for the specific purpose of increasing the overall positive charge of the dye. The dyes have been made as carboxylic acids to enable their use in labelling DNA or other biological molecules via active ester derivatives. The increased positive charge may be beneficial in electrostatic interactions with DNA in certain specific applications and in providing labelled nucleotides having particular charges for other purposes. It is also envisaged that at least one sulphonic acid group can be added to any of the +3 (or more) dyes to give a dye that may have an overall positive or negative charge or may be neutral and may have improved photostability and brightness. This improvement is useful in applications such as difference gel electrophoresis technology as described in WO 96/33406 where the overall charge on the dye is of importance.
The carboxylic acid derivative can be reacted: either with diamine species such as 1,3-diaminopropane or ethylene diamine to provide a primary amine functional group; or with a protected hydrazine to generate a corresponding hydrazide which can be deprotected; e.g. for linking to carbohydrates. The addition of extra quaternary amino groups and the controlled use of sulphonic acid groups can lead to a range of dyes having overall positive charges ranging up to 6 or even more.
Cy3 (n=1) and Cy5 (n=2) and Cy7 (n=3) dyes have the added advantage of allowing multiplexing i.e. the use of mixtures of targets labelled with different dyes for simultaneous analysis. This concept can also be increased by varying the intermediate derivative between indole, thiazole and oxazole derivatives, and by altering the number of fused aromatic rings to the dye.
This section shows the chemistry envisaged in making cyanine dyes having positive charges from 2 to 6, most but not all of which fall within the scope of the claims. Each numbered paragraph starts with a general picture of a cyanine dye shown as a rectangle, having a single positive charge shown as + within a circle. To two corners of the rectangle are attached curved lines which may comprise at least one positive charge and/or at least one functional or reactive group Q or Qxe2x80x2; these curved lines correspond to R1, R2, R3, R4, R5, R6 and R7, most usually R1 and R2, in the structures (1) and (2) shown above and in the claims. Some of the cyanine dyes have been made and are described below in the Examples; others are in preparation or are envisaged.
1. 
The dye carries an inherent +1 charge. A second + charge is located on a chain attached to one of the dye N atoms. A functional or reactive group Q terminates a chain attached to the other dye N atom.